1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosine

ABSTRACT

The present invention relates to 1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosines having excellent antitumor activity, represented by formula [I]: ##STR1## wherein R represents a hydrogen atom or a phosphoric acid residue, and to a process for the production and use thereof.

This application is a 371 of PCT/JP97/01206 filed Apr. 9, 1997.

TECHNICAL FIELD

The present invention relates to novel1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosines and aprocess for the production and use thereof.

BACKGROUND ART

A research group of University of Birmingham makes reference to1-(2-fluoro-4-thio-beta-D-arabinofuranosyl)-5-methyluracil and1-(2-fluoro-4-thio-beta-D-arabinofuranosyl)-5-iodocytosine inInternational Patent Application PCT/GB90/01518 (InternationalPublication Number: WO 91/04982).

Further, a research group of NIH has recently made a report on suchcompounds as1-(2,3-dideoxy-2-fluoro-4-thio-beta-D-erythro-pentafuranosyl)uracilrepresented by the following formula: ##STR2## provided that B is uracilin series a, and cytosine in series b (Tetrahedron Letters, 35,7569-7572 (1994); Tetrahedron Letters, 35, 7573-7576 (1994); ChemistryLetters, 301-302 (1995)).

However, the group of University of Birmingham describes the above twocompounds as mere instances, and fails to show examples in which theywere practically synthesized. Moreover, although a process for producingthe compounds is explained in the specification for the aforementionedinternational patent application, the process is simply an applicationof those processes which are described in known references (J. Org.Chem., 50, 2597 (1985), J. Org. Chem., 50, 3644 (1985)). It is clearfrom the above-described reports made by the group of NIH that desiredcompounds can never be obtained by employing such a process. Thespecification for the foregoing international application slightly makesmention of the antiviral activity of the compounds, but is quite silenton specific data in terms of the activity.

Further, although the group of NIH makes reference to the anti-HIVactivity of the compounds synthesized, the activity is not necessarilysatisfactory.

Furthermore, both of the groups have made no report on other biologicalactivities than antiviral activity.

DISCLOSURE OF THE INVENTION

We formerly developed a simple process for synthesizing2'-deoxy-2'-substituted-4'-thionucleoside derivatives, using glucose asa starting material (WO 96/01834). After this, we continued our studieson the basis of knowledge acquired in the course of the development ofthis process. As a result, we have established a simple process forsynthesizing 2'-deoxy-2'-fluoro-4'-thioarabinonucleosides, andsynthesized various compounds by using this new process. In the processof testing the biological activities of the compounds synthesized, wehave found that especially1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosines haveexcellent antitumor activity. The present invention has beenaccomplished on the basis of this finding.

The present invention therefore relates to1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosines representedby the following formula [I]: ##STR3## wherein R represents a hydrogenatom or a phosphoric acid residue, and to pharmaceutical compositions,especially antitumor agents, comprising these compounds as activeingredients.

Further, the present invention also relates to a process for producing1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosines representedby the above formula [I], comprising the following 1st to 3rd steps:

1st step:

a step of reacting a compound represented by formula [II] withdiethylaminosulfur trifluoride (DAST) after protecting the primaryhydroxyl group of the compound [II], thereby obtaining a compoundrepresented by formula [III]: ##STR4## wherein R₁ and R₂ represent analkyl, silyl or acyl group;

2nd step:

a step of converting the compound represented by formula [III] into asulfoxide by reacting the compound [III] with an oxidizing agent, andsubjecting the sulfoxide to Pummerer rearrangement reaction by treatingit with an acid anhydride or acid chloride, thereby obtaining a compoundrepresented by formula [IV]: ##STR5## wherein R₁ and R₂ are as definedabove, and R₃ represents an acyl group; and

3rd step:

a step of subjecting the compound represented by formula [IV] and N⁴-acylcytosine or cytosine to glycosylation reaction in the presence of aLewis acid catalyst to obtain a protected compound, removing theprotecting groups, and, if desired, phosphorylating the 5'-position ofthe sugar moiety of the compound, thereby obtaining a1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosine representedby formula [I]: ##STR6## wherein R, R₁, R₂ and R₃ are as defined above.

Furthermore, the present invention relates to a process for producing1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosines representedby the above formula [I], comprising the following 1st to 4th steps:

1st step:

a step of introducing a leaving group into the hydroxyl group on acompound represented by formula [V], and treating the compound with anucleophilic reagent by which a fluorine atom can be introduced, therebyobtaining a compound represented by formula [VI]: ##STR7##

2nd step:

a step of selectively deprotecting the isopropylidene group at the 5-and 6-positions of the compound represented by formula [VI], selectivelyprotecting the primary hydroxyl group of the compound, introducing aleaving group into the secondary hydroxyl group of the compound,deprotecting the primary hydroxyl group, performing 5,6-epoxidation,performing 5,6-thiiranation by using a sulfurizing reagent, opening thethiirane ring by using a nucleophilic reagent, and causing acylation,thereby obtaining a compound represented by formula [VII]: ##STR8##wherein R₄ and R₅ represent an alkyl or acyl group;

3rd step:

a step of hydrolyzing the isopropylidene group at the 1- and 2-positionsof the compound represented by formula [VII], carrying out oxidation byusing an oxidizing agent, alkoxylating the 1-position of the compound,and protecting the hydroxyl group with a protecting group, therebyobtaining a compound represented by formula [VIII]: ##STR9## wherein R₄and R₅ are as defined above, R₆ and R₇ represent an alkyl or acyl group,and R₈ represents an alkyl group; and

4th step:

a step of brominating the alkoxy group at the 1-position of the compoundrepresented by formula [VIII] by treating the compound [VIII] with ahydrogen bromide-acetic acid solution, subjecting the brominatedcompound and activated cytosine to glycosylation reaction to obtain aprotected compound, removing the protecting groups, and, if desired,phosphorylating the 5'-position of the sugar moiety of the compound,thereby obtaining a1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosine representedby formula [I]: ##STR10## wherein R₆, R₇ and R₈ are as defined above,and R represents a hydrogen atom or a phosphoric acid residue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the in vivo antitumor effect of the compoundof the present invention on the human colonic carcinoma SW-48 cells,obtainable when the compound is administered intravenously.

FIG. 2 is a graph showing the in vivo antitumor effect of the compoundof the present invention on the human colonic carcinoma SW-48 cells,obtainable when the compound is administered orally.

BEST MODE FOR CARRYING OUT THE INVENTION

(1) Compounds of the Invention

The compounds of the present invention are1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosines representedby the above formula [I]. These compounds may also be in the form ofsalt, hydrate or solvate. Examples of the salts include pharmaceuticallyacceptable salts, for example, acid adducts such as hydrochlorides andsulfates when R is a hydrogen atom; and alkaline metal salts such assodium, potassium and lithium salts, alkaline earth metal salts such asa calcium salt, and ammonium salts when R is a phosphoric acid residue.

Examples of the hydrates or solvates include those which are obtained byattaching 0.1 to 3.0 molecules of water or a solvent to one molecule ofthe compounds of the present invention or salts thereof. Further,various isomers such as tautomers can also be included in the compoundsof the present invention.

(2) Process for Producing the Compounds of the Invention

The compounds of the present invention can be produced by a reactionprocess comprising the following 1st to 3rd steps.

1st step:

The 1st step is a step of reacting a compound represented by formula[II] with DAST after protecting the primary hydroxyl group of thecompound [II], thereby obtaining a compound represented by formula[III]: ##STR11## wherein R₁ and R₂ represent an alkyl, silyl or acylgroup.

The starting compound is represented by the above formula [II], and canbe readily synthesized from glucose by a known method (J. Org. Chem.,61, 822 (1996)).

R₁ and R₂ in the formula are as defined above. Specific examples of R₁or R₂ include unsubstituted or substituted alkyl groups such as methyl,ethyl, benzyl, methoxybenzyl, dimethoxybenzyl, trityl anddimethoxytrityl, unsubstituted or substituted silyl groups such ast-butyldimethylsilyl and t-butyldiphenylsilyl, and acyl groups such asacetyl, benzoyl and pivaloyl.

The introduction of the protecting group can be achieved by aconventional technique. For example, in the case where a silylprotecting group is introduced, the reaction may be carried out by using1 to 10 moles of a silylating agent (e.g., t-butyldiphenylsilylchloride, t-butyldimethylsilyl chloride, or the like), and, whennecessary, 1 to 5 moles of a base such as imidazole for 1 mole of thecompound represented by formula [II], in a reaction solvent (e.g., asingle solvent or solvent mixture of pyridine, picoline,dimethylaminiopyridine, dimethylformamide, acetonitrile, methylenechloride, or the like) at a temperature of 0 to 50° C.

The compound having a protecting group thus obtained is allowed to reactwith DAST to obtain a compound of formula [III].

The reaction with DAST can be carried out by using 1 to 20 moles of DASTfor 1 mole of the compound of formula [II], in a solvent such asmethylene chloride, dichloroethane, benzene, toluene, xylene, hexane orthe like, at a temperature of -100 to 150° C., preferably -80° C. toroom temperature, if necessary, in an atmosphere of an inert gas such asargon or nitrogen.

The compound of formula [III] may be isolated by a conventional meansfor the isolation and purification of sugar. For instance, it ispossible to purify the reaction solution by silica gel columnchromatography after it is partitioned between ethyl acetate and water,thereby isolating the compound [III].

2nd step:

The 2nd step is a step of converting the compound represented by formula[III] into a sulfoxide by reacting the compound [III] with an oxidizingagent, and subjecting the sulfoxide to Pummerer rearrangement reactionby treating it with an acid anhydride or acid chloride, therebyobtaining a compound represented by formula [IV]: ##STR12## wherein R₁and R₂ are as defined above, and R₃ represents an acyl group.

The derivation to a sulfoxide can be achieved by a conventional method.For instance, a method in which a compound is treated withm-chloroperbenzoic acid in methylene chloride at a temperature of -100to 0° C. under a stream of an inert gas such as argon or nitrogen (J.Org. Chem., 61, 822 (1996)), or a method in which a compound is treatedwith sodium metaperiodate in an alcohol solvent such as methanol(Tetrahedron Letter, 993 (1979)) can be utilized.

The Pummerer rearrangement reaction, which is performed by a treatmentwith an acid anhydride or acid chloride, can also be carried out by aconventional method. Namely, the reaction can be carried out by using anacid anhydride such as acetic anhydride or trifluoroacetic anhydride, oran acid chloride such as mesyl chloride in an amount of 1 to 100 molesfor 1 mole of the sulfoxide, at a temperature of -80 to 150° C., whennecessary, under a stream of an inert gas such as argon or nitrogen.Although the acid anhydride or acid chloride used functions as areaction solvent, it is also possible to carry out the above reaction inan organic solvent such as methylene chloride, when necessary.

The compound of formula [IV] thus obtained may be isolated by aconventional means for the isolation and purification of sugar. Forinstance, it is possible to purify the reaction solution by silica gelcolumn chromatography after it is partitioned between ethyl acetate andwater, thereby isolating the compound [IV].

3rd step:

The 3rd step is a step of subjecting the compound represented by formula[IV] and N⁴ -acylcytosine or cytosine to glycosylation reaction in thepresence of a Lewis acid catalyst to obtain a protected compound,removing the protecting groups, and, if desired, phosphorylating the5'-position of the sugar moiety of the compound, thereby obtaining a1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosine representedby formula [I]: ##STR13## wherein R₁, R₂, R₃, and R are as definedbefore.

The glycosylation reaction of the compound of formula [IV] can becarried out by using 1 to 10 moles of N⁴ -acylcytosine or cytosine and0.1 to 10 moles of a Lewis acid such as trimethylsilyltrifluoromethanesulfonate, tin tetrachloride, titanium tetrachloride,zinc chloride or boron trifluoride for 1 mole of the compound of formula[IV], in a solvent such as methylene chloride, chloroform,dichloroethane, acetonitrile or dimethylfor-mamide, at a temperature of-50 to 100° C., when necessary, under a stream of an inert gas such asargon or nitrogen.

The removal of the protecting group may be attained by a techniqueproperly selected, depending upon the type of the protecting group used,from conventional techniques such as acid hydrolysis, alkali hydrolysis,a treatment with tetrabutylammonium fluoride, and catalytichydrogenation For example, in the case where a benzyl protecting groupis removed, deprotection can be attained by a method in which thecompound is treated with boron trichloride or boron tribromide inmethylene chloride at a temperature of -100° C. to room temperatureunder a stream of an inert gas such as argon or nitrogen.

Further, when a compound of formula [I] in which R is a phosphoric acidresidue is synthesized, the desired compound of free acid type or salttype can be obtained by a conventional method, by reacting the compoundwhich has been deprotected by the above method with a phosphorylatingagent used for a conventional reaction for selectively phosphorylatingthe 5'-position of nucleosides, such as phosphorus oxychloride,tetrachloropyrophosphoric acid or beta-cyanoethylphosphoric acid-DCC.

The compound of the present invention thus obtained can be isolated andpurified by a technique which is a proper combination of techniquesconventionally used for the isolation and purification of nucleosides ornucleotides. For instance, in the case of the isolation of a nucleosidederivative (where R in formula [I] is a hydrogen atom), the desiredcompound may be obtained by crystallization from a proper solvent suchas ethanol, which is carried out after the solvent in the reactionsolution is distilled off. The desired compound can also be obtained asa salt-type compound, if necessary. Further, in the case of theisolation of a nucleotide derivative (where R in formula [I] is aphosphoric acid residue), the reaction solution may be purified byion-exchange column chromatography, or adsorption column chromatographyusing activated carbon or the like, and then freeze-dried orcrystallized to obtain the desired compound of free acid type. Ifnecessary, the desired compound can also be obtained as a salt-typecompound.

Alternatively, the compounds of the present invention can also beproduced by a process comprising the following 1st to 4th steps. Thisprocess is advantageous in that the reaction conditions to be employedare relatively mild and that improvements in the reaction yield and inthe yield of a beta-derivative can be attained or expected.

1st step:

The 1st step is a step of introducing a leaving group into the hydroxylgroup on a compound represented by formula [V], and treating thecompound with a nucleophilic reagent by which a fluorine atom can beintroduced, thereby obtaining a compound represented by formula [VI]:##STR14##

The starting compound is represented by formula [V], and can be readilyproduced from glucose by a known method (Carbohydr. Res., 24, 192(1972)).

Examples of the leaving group to be introduced include sulfonyl groupssuch as methanesulfonyl, p-toluenesulfonyl, benzenesulfonyl,imidazoylsulfonyl and trifluoromethanesulfonyl. Methanesulfonyl,p-toluenesulfonyl and imidazoylsulfonyl are preferred.

The introduction of the leaving group may be achieved by a conventionaltechnique. For instance, in the case where methanesulfonyl,p-toluenesulfonyl or imidazoyl sulfonyl group is introduced, thereaction can be carried out by using 1 to 10 moles of methanesulfonylchloride, p-toluenesulfonyl chloride or sulfuryl chloride, and, whennecessary, 2 to 50 moles of a base such as imidazole for 1 mole of thecompound of formula [V], in a reaction solvent (e.g., a single solventor solvent mixture of pyridine, picoline, dimethylaminopyridine,dimethylformamide, acetonitrile, methylene chloride or the like), at atemperature of -50 to 50° C., preferably 0 to 50° C.

As the nucleophilic reagent by which a fluorine atom can be introduced,potassium fluoride (including a spray-dried product), potassiumhydrogenfluoride, ammonium fluoride, ammonium hydrogenfluoride,tetrabutylammonium fluoride or the like can be used.

The reaction with such a nucleophilic reagent may be carried out byusing a nucleophilic reagent in an amount of 2 to 100 moles, preferably2 to 50 moles for 1 mole of the compound of formula [V], in a glycolsolvent such as 2-methoxyethanol or 2,3-butanediol, at a temperatureranging from room temperature to 300° C., preferably from 50 to 200° C.

The compound of formula [VI] thus obtained may be isolated by aconventional means for the isolation and purification of sugar. Forinstance, it is possible to purify the reaction solution by silica gelcolumn chromatography after it is partitioned between ethyl acetate andwater, followed by elution with an organic solvent such asn-hexane/ethyl acetate, thereby isolating the compound [VI].

2nd step:

The 2nd step is a step of selectively deprotecting the isopropylidenegroup at the 5- and 6-positions of the compound represented by formula[VI], selectively protecting the primary hydroxyl group of the compound,introducing a leaving group into the secondary hydroxyl group of thecompound, deprotecting the primary hydroxyl group, performing5,6-epoxidation, performing 5,6-thiiranation by using a sulfurizingreagent, opening the thiirane ring by using a nucleophilic reagent, andcausing acylation, thereby obtaining a compound represented by formula[VII]: ##STR15## wherein R₄ and R₅ represent an alkyl or acyl group.

The selective deprotection of the isopropylidene group at the 5- and6-positions may be achieved by a conventional technique of acidhydrolysis. Examples of acids that can be used in the acid hydrolysisinclude mineral acids such as hydrochloric acid and sulfuric acid, andorganic acids such as acetic acid, trifluoroacetic acid andp-toluenesulfonic acid. When the deprotection reaction is carried out,an acid to be used is diluted with water to a proper concentration, and,when necessary, the diluted acid is mixed with an organic solvent suchas THF or dioxane to obtain a solvent mixture. The deprotection reactioncan be carried out by using such an acid at a temperature of -50 to 150°C., preferably -20 to 100° C., with stirring.

An ordinary hydroxy-protecting group may be used as the protecting groupwhich is used for the selective protection of the primary hydroxylgroup. Examples of such a protecting group include benzyl protectinggroups such as benzyl and dimethoxybenzyl, silyl protecting groups suchas t-butyldimethylsilyl, t-butyldiphenylsilyl and triethylsilyl, etherprotecting groups such as methoxymethyl, methoxyethoxyethyl,tetrahydrofuran and tetrahydropyran, trityl protecting groups such astrityl, monomethoxytrityl, dimethoxytrityl, and acyl groups such asacetyl, benzoyl and pivaloyl.

The introduction of such a protecting group may be achieved by aconventional means. For example, in the case where a silyl protectinggroup such as t-butyldiphenylsilyl group, or an acyl group such asbenzoyl group is introduced, the reaction can be carried out by using0.8 to 10 moles of a silylating agent (e.g., t-butyldiphenylsilylchloride, or the like) or acylating agent (e.g., benzoyl chloride, orthe like), and, when necessary, 1 to 5 moles of a base such as imidazoleor pyridine for 1 mole of the compound of formula [VI], in a reactionsolvent (e.g., a single solvent or solvent mixture of pyridine,picoline, dimethylaminopyridine, dimethylformamide, acetonitrile,methylene chloride, or the like), at a temperature of -20 to 50° C.

Further, as the leaving group to be introduced into the secondaryhydroxyl group, the same leaving groups as those enumerated in the 1ststep can be used. The introduction of such a leaving group can beeffected by the same method as that described in the 1st step.

The deprotection of the primary hydroxyl group may be achieved by atechnique properly selected, depending upon the protecting group used,from conventional techniques such as acid hydrolysis, alkali hydrolysiscombined with ester interchange, a treatment with a fluoride, andcatalytic hydrogenation. In particular, when alkali hydrolysis and esterinterchange are performed, epoxidation reaction also proceedssimultaneously under the same conditions. However, when the epoxidationreaction proceeded merely insufficiently under the conditions of thealkali hydrolysis and ester interchange, or when the deprotection wasconducted under other conditions, the cis-diol derivative obtained bythe deprotection can be converted into the desired epoxy derivative bytreating the cis-diol derivative with a base. Examples of the bases thatcan be used in this treatment include sodium hydride, potassium hydride,butyl lithium, lithium diisopropylamide, sodium methoxide, sodiumethoxide, potassium carbonate, and sodium carbonate. The treatment withsuch a base can be carried out by using 0.5 to 5 moles of a base for 1mole of the compound of formula [IV], in an organic solvent such as anether solvent, for instance, ether, THF or dioxane, or an alcoholsolvent, for instance, methanol or ethanol, at a temperature of -50 to120° C.

The conversion of the epoxidized derivative obtained into a thuiiranederivative can be effected by using 0.1 to 10 moles of a sulfurizingreagent for 1 mole of the compound of formula [VI], in an organicsolvent such as an alcohol solvent (for example, methanol, ethanol orisopropano), pyridine, acetonitrile or DMF, at a temperature of 0 to150° C. Examples of sulfurizing reagents that can be used in the abovetreatment include thiourea, xanthate and thiocarbonyl diimidazole.

The ring opening of the thiirane derivative obtained, and theintroduction of an acyl group can be effected by using 1 to 100 moles ofan organic acid, organic acid salt or acid anhydride for 1 mole of thecompound of formula [VI], in any mixture of organic acids, organic acidsalts and acid anhydrides, at a temperature ranging from roomtemperature to 200° C. Examples of organic acids that can be used in theabove reaction include acetic acid, propionic acid, benzoic acid,pivalic acid and trifluoroacetic acid; examples of organic acid saltsinclude sodium acetate, potassium acetate, lithium acetate, sodiumpropionate, potassium propionate, lithium propionate, sodium benzoate,potassium benzoate, lithium benzoate, sodium trifluoroacetate, potassiumtrifluoroacetate and lithium trifluoroacetate; and examples of acidanhydrides include acetic anhydride, propionic anhydride, benzoicanhydride, pivalic anhydride and trifluoroacetic anhydride.

The compound of formula [VII] thus obtained may be isolated by aconventional means for the isolation and purification of sugar. Forinstance, it is possible to purify the reaction solution by silica gelcolumn chromatography after it is partitioned between ethyl acetate andwater, followed by elution with an organic solvent such asn-hexane/ethyl acetate, thereby isolating the compound [VII].

3rd step:

The 3rd step is a step of hydrolyzing the isopropylidene group at the 1-and 2-positions of the compound represented by formula [VII], carryingout oxidation by using an oxidizing agent, alkoxylating the 1-position,and protecting the hydroxyl group with a protecting group, therebyobtaining a compound represented by formula [VIII]: ##STR16## wherein R₄and R₅ are as defined before, R₆ and R₇ represent an alkyl or acylgroup, and R₈ represents an alkyl group.

The hydrolysis of the isopropylidene group at the 1- and 2-positions canbe carried out by the same method as that used for the deprotection ofthe isopropylidene group at the 5- and 6-positions, described in theabove 2nd step.

The oxidation can be carried out by using 0.1 to 10 moles of anoxidizing agent such as sodium periodate or potassium permanganate for 1mole of the compound of formula [VII], in a single solvent of an organicsolvent such as an alcohol solvent (for example, methanol), an ethersolvent (for example, THF or dioxane), methylene chloride,dichloroethane, benzene or toluene, or in a solvent mixture of such asolvent with water, at a temperature of -50 to 100° C., preferably -20to 50° C.

The alkoxylation reaction of the 1-position can be effected by usinglargely excessive hydrogen chloride, in an alcohol solvent such asmethanol, ethanol, isopropanol, t-butanol or benzyl alcohol, at atemperature of -50 to 100° C.

An ordinary hydroxy-protecting group may be used as the protecting groupfor the hydroxyl groups at the 3- and 5-positions. Examples of such aprotecting group include benzyl protecting groups such as benzyl anddimethoxybenzyl, silyl protecting groups such as t-butyldimethylsilyl,t-butyldiphenylsilyl and triethylsilyl, ether protecting groups such asmethoxymethyl, methoxyethoxyethyl, tetrahydrofuran and tetrahydropyran,trityl protecting groups such as trityl, monomethoxytrityl anddimethoxytrityl, and acyl groups such as acetyl, benzoyl and pivaloyl.The introduction of a protecting group can be achieved by the samemethod as that described in the above 2nd step.

The compound of formula [VIII] thus obtained may be isolated by aconventional means for the isolation and purification of sugar. Forexample, it is possible to purify the reaction solution by silica gelcolumn chromatography after it is partitioned between ethyl acetate andwater, followed by elution with an organic solvent such asn-hexane/ethyl acetate, thereby isolating the compound [VIII].

4th step:

The 4th step is a step of brominating the alkoxy group at the 1-positionof the compound represented by formula [VIII] by treating the compound[VIII] with a hydrogen bromide/acetic acid solution, subjecting thebrominated compound and activated cytosine to glycosylation reaction toobtain a protected compound, removing the protecting groups, and, ifdesired, phosphorylating the 5'-position of the sugar moiety of thecompound, thereby obtaining a1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosine representedby formula [I]: ##STR17## wherein R₆, R₇ and R₈ are as defined before,and R represents a hydrogen atom or a phosphoric acid residue.

The bromination of the alkoxy group at the 1-position of the compound offormula [VIII] can be effected by treating the compound with a hydrogenbromide-acetic acid solution containing approximately 0.1 to 10 moles ofhydrogen bromide for 1 mole of the compound of formula [VIII], with orwithout a solvent mixture with methylene chloride, chloroform,dichloroethane or the like, at a temperature of -50 to 70° C.

Further, in the case where the above bromination reaction does not fullyproceed, it is possible to firstly decompose the compound of formula[VIII] by adding acetic acid to obtain a 1-acetoxy derivative, which maythen be subjected to the above-described bromination reaction. Thedecomposition of the compound of formula [VIII] with the addition ofacetic acid is effected in a mixture of acetic acid and acetic anhydridein an amount of 1 mole to a largely excessive amount for 1 mole of thecompound of formula [VIII], in the presence of a mineral acid such assulfuric acid, at a temperature of -20 to 100° C., preferably 0 to 50°C.

The glycosylation reaction can be carried out by using 1 to 10 moles ofactivated cytosine (silylated cytosine, or a metallic salt or alkylammonium salt of cytosine), and, when necessary, 0.1 to 10 moles of aLewis acid such as trimethylsilyl trifluoromethanesulfonate, tintetrachloride, titanium tetrachloride, zinc chloride or borontrifluoride for 1 mole of the compound of formula [VIII], in a reactionsolvent such as methylene chloride, chloroform, dichloroethane,acetonitrile, dimethylformamide or the like, at a temperature of -50 to100° C., under a stream of an inert gas such as argon or nitrogen.

Alternatively, it is also possible to carry out the glycosylationreaction in an organic solvent such as methylene chloride, chloroform,dichloroethane or acetonitrile, or in the absence of a solvent, in thepresence of silylated cytosine, and, when necessary, a catalyst such assodium iodide, at a temperature ranging from room temperature to 200° C.

The protecting group may be removed by a technique properly selected,depending upon the protecting group used, from conventional techniquessuch as acid hydrolysis, alkali hydrolysis, a treatment with a fluoride,and catalytic hydrogenation. In particular, in the case where a benzylprotecting group is removed, it is desirable to employ a method in whichdeprotection is achieved by using boron trichloride or boron tribromidein methylene chloride at a temperature ranging from -100° C. to roomtemperature under a stream of an inert gas such as argon or nitrogen.

Further, when a compound of formula [I] in which R is a phosphoric acidresidue is synthesized, the desired compound of free acid type or salttype can be obtained by a conventional method, e.g., by reacting thecompound which has been produced and deprotected by the above methodwith a phosphorylating agent used for a conventional reaction forselectively phosphorylating the 5'-position of nucleosides, such asphosphorus oxychloride, tetrachloropyrophosphoric acid orbeta-cyanoethylphosphoric aci-DCC.

The compound of the present invention thus obtained can be isolated andpurified by a technique which is a proper combination of techniquesconventionally used for the isolation and purification of nucleosides ornucleotides. For instance, in the case of the isolation of a nucleosidederivative (where R in formula [I] is a hydrogen atom), the desiredcompound may be obtained by crystallization from a proper solvent suchas ethanol, which is carried out after the solvent in the reactionsolution is distilled off. The desired compound can also be obtained asa salt-type compound, if necessary. Further, in the case of theisolation of a nucleotide derivative (where R in formula [I] is aphosphoric acid residue), the reaction solution may be purified byion-exchange column chromatography, or adsorption column chromatographyusing activated carbon or the like, and then freeze-dried orcrystallized to obtain the desired compound of free acid type. Ifnecessary, the desired compound can also be obtained as a salt-typecompound.

(3) Use of the Compounds of the Invention

The compounds of the present invention have excellent antitumor activityas shown in Test Examples, which will be described later. Therefore, thecompositions of the present invention comprising these compounds asactive ingredients are useful in the treatment of malignant tumors(e.g., lung cancer, carcinoma of the esophagus, gastric cancer, coloniccancer, rectal cancer, pancreatic carcinoma, breast cancer, cancer ofthe kidney, bladder carcinoma, carcinoma uteri, osteosarcoma andmelanoma).

The dose of the compounds of the present invention varies depending uponthe age and body weight of the recipient, the disease, the severity ofthe condition of the recipient, the permissibility, and the route foradministration; and it is to be properly decided by taking all of thesefactors into consideration. In general, however, the dose is selectedfrom the range of 0.001 to 1,000 mg per kilogram body weight, preferablyfrom the range of 0.01 to 100 mg per kilogram body weight. The desireddose is administered at one time. Alternatively, the desired dose isdivided into sub-doses, and the sub-doses are administered several timesper day.

The compounds can be administered via any route; they can beadministered orally, parenterally, rectally or topically. When thecompounds of the present invention are made into formulations, carriers,excipients and other additives which are usually used for conventionalformulations can be used. Examples of the carriers include solidcarriers such as lactose, kaolin, sucrose, crystalline cellulose, cornstarch, talc, agar, pectin, stearic acid, magnesium stearate, lecithinand sodium chloride, and liquid carriers such as glycerin, peanut oil,polyvinyl pyrrolidone, olive oil, ethanol, benzyl alcohol, propyleneglycol and water.

The formulations may be presented in any form. For instance, when asolid carrier is used, the form includes tablet, powder, granule,capsule, suppository and troche; and, when a liquid carrier is used, theform includes syrup, emulsion, soft gelatin capsule, cream, gel, paste,spray and injection.

EXAMPLES

The present invention will now be specifically explained by referring tothe following Synthesis Examples, Test Examples and FormulationExamples. However, the present invention is not limited by theseexamples in any way.

Synthesis Example 1 Synthesis of1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosine (R=H informula [I])

(1) Synthesis of1,4-anhydro-5-O-t-butyldiphenylsilyl-3-O-benzyl-2-deoxy-2-fluoro-4-thio-D-arabitol(R₁ =Bn and R₂ =TBDPS in formula [III])

37.7 g of 1,4-anhydro-3-O-benzyl-4-thio-D-arabitol (R₁ =Bn in formula[II]) and 11.3 g of imidazole were dissolved in 400 ml of DMF. To thissolution was added 42.9 ml of t-butyldiphenylsilyl chloride (TBDPSCl)with ice-cooling, and the mixture was stirred at 0° C. overnight under astream of argon. Water was added to the mixture, and the resultingmixture was stirred at room temperature for a while. Thereafter, thesolvent was distilled off, and the residue was partitioned between ethylacetate and water. The organic layer was further washed with water, anddried. The solvent was concentrated, and the residue was purified bysilica gel column chromatography. The fractions eluted with 2-10% ethylacetate/n-hexane were concentrated to obtain 53.4 g (yield 71%) of the5-silyl derivative.

5.06 g of the 5-silyl derivative was dissolved in 25 ml of methylenechloride. To this solution, 25 ml of a methylene chloride solutioncontaining 2.26 ml of diethylaminosulfur trifluoride (DAST) was addeddropwise at -78° C. under a stream of argon, and the mixture was stirredat -78° C. for 3 hours. The reaction was terminated by adding asaturated aqueous solution of sodium hydrogencarbonate, and the reactionsolution was extracted with chloroform. The organic layer was dried overanhydrous sodium sulfate, and the solvent was distilled off. The residuewas purified by silica gel column chromatography. The fractions elutedwith 2-4% ethyl acetate/n-hexane were concentrated to obtain 2.78 g(yield 55%) of the desired product.

¹ H-NMR (CDCl₃) δ 7.71-7.63 (4H, m, C₆ H₅) 7.71-7.63 (11H, m, C₆ H₅),5.18 (1H, d q, H-2, J=3.5, 50.5 Hz), 4.64 (1H, d, C₆ H₅ CH₂, J=12.0 Hz),4.60 (1H, d, C₆ H₅ CH₂, J=12.0 Hz), 4.35 (1H, dt, H-3, J=2.9, 11.2 Hz),3.76 (1H, t, H-5a, J=9.5 Hz), 3.66 (1H, ddd, H-5b, J=2.0, 6.1, 10.5 Hz),3.57-3.53 (1H, m, H-4) 3.19 (1H, ddd, H-1a, J=4.4, 12.2, 30.3 Hz) 3.06(1H, ddd, H-1b, J=3.4, 12.2, 18.1 Hz), 1.05 (9H, s, tBu)

(2) Synthesis of1-O-acetyl-5-O-t-butyldiphenylsilyl-3-O-benzyl-2-deoxy-2-fluoro-4-thio-D-arabinose(R₁ =Bn, R₂ =TBDPS and R₃ =Ac in formula [IV])

2.58 g of the1,4-anhydro-5-O-t-butyldiphenylsilyl-3-O-benzyl-2-deoxy-2-fluoro-4-thio-D-arabitolwas dissolved in 15 ml of methylene chloride, and the solution wascooled to -78° C. under a stream of argon. To this solution, a solutionof 1.15 g of 80% m-chloroperbenzoic acid in methylene chloride was addeddropwise, and the mixture was stirred for 30 minutes. Thereafter, thereaction was terminated by adding a saturated sodium hydrogencarbonatesolution. The reaction solution was allowed to warm up to roomtemperature, and extracted with chloroform. The organic layer was dried,and the solvent was distilled off. The residue was dissolved in 30 ml ofacetic anhydride, and the temperature of the solution was maintained at110° C. for 2 hours under a stream of argon. After the solution wascooled to room temperature, the solvent was distilled off under reducedpressure. The residue was dissolved in ethyl acetate, and the solutionwas partitioned among water, a saturated aqueous solution of sodiumhydrogencarbonate, and a saturated saline solution, and then dried overanhydrous sodium sulfate. The solution was concentrated under reducedpressure, and the residue was purified by silica gel columnchromatography. The fractions eluted with 5-10% ethyl acetate/n-hexanewere concentrated to obtain 1.57 g (yield 54%) of the desired product.

¹ H-NMR (CDCl₃) δ 7.68-7.62 (4H, m, C₆ H₅) 7.46-7.25 (11H, m, C₆ H₅)6.06 (1H, d, H-1, J=4.4 Hz), 5.11 (1H, ddd, H-2, J=4.4, 8.3, 51.0 Hz),4.78 (1H, d, C₆ H₅ CH₂, J=11.7 Hz), 4.60 (1H, d, C₆ H₅ CH₂, J=11.7 Hz),4.38 (1H, ddd, H-3, J=7.3, 8.3, 11.7 Hz), 3.81 (1H, dd, H-5a, J=4.4,10.5 Hz), 3.74 (1H, dd, H-5b, J=5.9, 10.5 Hz), 3.34 (1H, ddd, H-4,J=4.4, 5.9, 7.3 Hz), 2.05 (3H, s, Ac), 1.07 (9H, s, tBu)

(3) Synthesis of 1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosine (R=H in formula [I])

971 mg of the1-O-acetyl-5-O-t-butyldiphenylsilyl-3-O-benzyl-2-deoxy-2-fluoro-4-thio-D-arabinosewas dissolved in 20 ml of acetonitrile. To this solution were addedsilylated N⁴ -acetylcytosine (prepared by heating 832 mg of N⁴-acetylcytosine and a catalytic amount of ammonium sulfate inhexamethyldisilazane for 5 hours under reflux), and 3.60 ml of asolution of 1M tin tetrachloride in methylene chloride and the mixturewas stirred at room temperature for 3 hours. To this mixture was furtheradded 1.80 ml of a solution of 1M tin tetrachloride in methylenechloride. The resulting mixture was stirred at room temperature for 1hour, and a saturated sodium hydrogencarbonate solution was then addedthereto. The mixture was filtered through Celite to remove insolubles,and extracted three times with chloroform. The organic layer was dried.The filtrate was concentrated under reduced pressure, and the residuewas purified by silica gel column chromatography. The fractions elutedwith 1% methanol/n-chloroform were collected, and concentrated to obtain672 mg (yield 59%) of the desired protected product.

614 mg of the protected product was dissolved in 15 ml of methylenechloride. To this solution, 4.85 ml of 1 M trichloroboran was addeddropwise at -78° C. under a stream of argon. The temperature of themixture was raised to 0° C., and the mixture was stirred for 30 minutes.To this mixture were added 2 ml of pyridine and 5 ml of methanol. Theresulting mixture was stirred at -78° C. for a further 30 minutes, andthen allowed to warm up to room temperature. The solvent was distilledoff, and the residue was azeotropically distilled three times withmethanol. The solvent was then distilled off, and the residue wasdissolved in 10 ml of methanol. To this solution was added 360 mg ofammonium fluoride, and the temperature of the mixture was maintained at60° C. for 2 hours. The mixture was then concentrated under reducedpressure. To the resultant were added 15 ml of methanol and 15 ml ofconcentrated aqueous ammonia, and the mixture was stirred at roomtemperature overnight. The solvent was distilled off, and the residuewas azeotropically distilled with ethanol. The residue was successivelypurified by silica gel column chromatography and ODS reverse phasecolumn chromatography to obtain 29 mg of the title compound.

¹ H-NMR (DMSO-d₆) δ 7.98 (1H, dd, H-6, J=1.0, 7.2 Hz) 7.26, 7.19 (2H,br, NH₂) 6.47 (1H, dd, H-1, J=5.4, 13.2 Hz) 5.85 (1H, d, 3-OH, J=4.9Hz), 5.77 (1H, d, H-5, J=7.3 Hz), 5.22 (1H, t, 5-OH, J=5.4 Hz), 4.91(1H, dt, H-2', J=5.4, 50.8 Hz), 4.25 (1H, ddt, H-3', J=4.9, 5.4, 11.2Hz), 3.72 (1H, dt, H-5'a, J=5.4, 11.2 Hz), 3.60 (1H, dt, H-5'b, J=5.9,11.2 Hz), 3.22 (1H, dt, H-4', J=5.4, 5.9 Hz)

Synthesis Example 2 Synthesis of1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosine (R=H informula [I])

(1) Synthesis of1,2:5,6-di-O-isopropylidene-3-deoxy-3-fluoro-alpha-D-glucofuranose(formula [VI])

20.0 g (76.84 mmol) of 1,2:5,6-di-O-isopropylidenealpha-D-allofuranose(formula [V]) was dissolved in 240 ml of CH₂ Cl₂. To this solution wasadded dropwise 12.35 ml (153.68 mmol) of SO₂ Cl₂ at 0° C., and themixture was stirred for 15 minutes. Thereafter, 52.3 g (768.40 mmol) ofimidazole was slowly added to the mixture with ice-cooling, and theresulting mixture was stirred at room temperature for 2 to 3 hours.After the reaction was terminated by adding saturated NaHCO₃, thereaction solution was extracted with CHCl₃. The organic layer was driedover Na₂ SO₄, and the solvent was distilled off. The residue wasdissolved in 240 ml of 2-methoxyethanol. To this solution was added44.64 g (768.40 mmol) of potassium fluoride (spray-dried product), andthe mixture was heated under reflux at 130° C. for 4 to 6 hours. Afterthe mixture was allowed to cool, the solvent was distilled off, and theresidue was partitioned between ethyl acetate and water. The organiclayer was successively washed with H₂ O×2 and brine, dried over Na₂ SO₄,and then evaporated to dryness under reduced pressure. Purification bycolumn chromatography on silica gel (400 cc, 5-20% AcOEt (in hexane))gave 12.98 g (49.49 mmol) of the desired product in a yield of 64%.

¹ H-NMR (CDCl₃) δ 5.95 (d, 1H, H-1, J₁,2 =3.9 Hz), 5.01 (dd, 1H, H-3,J₃,4 =2.2 Hz, J₃,F =49.8 Hz), 4.70 (dd, H, H-2, J₁,2 =3.9 Hz, J₂,F =10.7Hz), 4.2 9 (1H, ddd, H-5, J₄,5 =8.3 Hz, J₅,6a =5.9 Hz, J₅,6b =4.9 Hz),4.12 (dd, 1H, H-6a, J₅,6a =5.9 Hz, J_(6a),b =8.8 Hz), 4.11 (ddd, 1H,H-4, J₃,4 =2.2 Hz, J₄.5 =8.3 Hz , J₄,F =29.0 Hz), 4.03 (dd, 1H, H-6b,J₅,6 =4.9 Hz, J_(6a),b =8.8 Hz) 1.50, 1.45, 1.37, 1.33 (s, each 3H, ipr)

(2) Synthesis of5,6-di-S,O-acetyl-1,2-O-isopropylidene-5-thio-alpha-D-glucofuranose (R₄=R₅ =Ac in formula [VII])

10.9 g (41.56 mmol) of1,2:5,6-di-O-isopropylidene-3-deoxy-3-fluoro-alpha-D-glucofuranose wasdissolved in 40 ml of THF and 40 ml of 2 N HCl, and the solution wasstirred at room temperature. After the reaction was completed, thereaction solution was neutralized with NaHCO₃, and the resultant wasfiltered to remove insolubles. The filtrate was extracted with CHCl₃.The organic layer was washed with brine, and dried over Na₂ SO₄. Thesolvent was distilled off, and the residue was purified by columnchromatography on silica gel (320 cc, 3-6% MeOH (in CHCl₃)) to obtain8.02 g (36.09 mmol) of1,2-O-isopropylidene-3-deoxy-3-fluoro-alpha-D-glucofuranose.1,2-O-Isopropylidene-3-deoxy-3-fluoroalpha-D-glucofuranose was dissolvedin 120 ml of CH₂ Cl₂. 3.20 ml of pyridine and 44 mg of DMAP were addedto the solution. To this mixture was added dropwise a solution of 4.61ml (39.68 mmol) of BzCl in 50 ml of CH₂ Cl₂ at -5° C. Reaction wascarried out at -5° C. for 5 hours. After the completion of the reactionwas confirmed, MeOH was added to the reaction solution, and the mixturewas stirred for 1 hour to terminate the reaction. The resultant waspartitioned between CHCl₃ and H₂ O. The organic layer was successivelywashed with 0.5 N HCl×2, saturated NaHCO₃ ×2, and brine, and then driedover Na₂ SO₄. The solvent was distilled off, and the residue waspurified by column chromatography on silica gel (320 cc, 10-25% AcOEt(in hexane)) to obtain 9.33 g (28.59 mmol) of6-O-benzoyl-1,2-O-isopropylidene-3-deoxy-3-fluoro-alpha-D-glucofuranosein a yield of 79%.

¹ H-NMR (CDCl₃) δ ppm 8.09-8.05 (m, 2H, Bz) 7.60-7.42 (m, 3H, Bz), 5.99(d, 1H, H-1, J₁,2 =3.9 Hz), 5.14 (dd, 1H, H-3, J₃,4 =2.0 Hz, J₃,F =49.8Hz), 4.74-4.70 (m, 2H, H-2, H-6a) 4.46 (dd, 1H, H-6b, J₅,6b =5.9 Hz,J_(6a),b =12.2 Hz 4.27-4.18 (m, 2H, H-4, H₅), 2.8 3 (br, 1H, 5-OH),1.47, 1.33 (s, each 3H, ipr)

9.33 g (28.59 mmol) of6-O-benzoyl-1,2-O-isopropylidene-3-deoxy-3-fluoro-alpha-D-glucofuranosewas dissolved in 80 ml of pyridine. To this solution was added dropwise3.32 ml (42.88 mmol) of MsCl at 0° C. and the mixture was stirred atroom temperature overnight. The reaction was terminated by adding H₂ Oat 0° C. and the reaction solution was extracted with ethyl acetate. Theorganic layer was washed with saturated NaHCO₃ and brine, and then driedover Na₂ SO₄. The solvent was distilled off. To the residue weresuccessively added 80 ml of MeOH and 7 ml (34.31 mmol) of 28% NaOCH₃.The mixture was stirred at room temperature for 45 minutes, and thenpartitioned between ethyl acetate and water. The organic layer was driedover Na₂ SO₄, and purified by column chromatography on silica gel (220cc, AcOEt:hexane=(5:1) to (1:1)) to obtain 3.82 g (65%) of5,6-anhydro-1,2-O-isopropylidene-3-deoxy-3-fluoro-alpha-L-idofuranose,and 1 .404 g (16 ) of1,2-O-isopropylidene-3-deoxy-3-fluoro-5-O-methanesulfonyl-alpha-D-glucofuranose.1,2-O-Isopropylidene-3-deoxy-3-fluoro-5-O-methanesulfonyl-alpha-D-glucofuranosewas dissolved in 10 ml of THF, and treated with 206 mg (5.14 mmol) of60% NaH to convert it into 5,6-anhydro-1,2-O-isopropylidene-3-deoxy-3-fluoro-alpha-L-idofuranose.

¹ H-NMR (CDCl₃) δ ppm 6.04 (d, 1H, H-1, J₁,2 =3.9 Hz), 4.96 (dd, 1H,H-3, J₃,4 =2.4 Hz, J₃,F =50.3 Hz), 4.71 (dd, 1H, H-2, J₁.2 =3.9 Hz, J₂,F=11.2 Hz), 3.89 (ddd, 1H, H-4, J₃,4 =2.4 Hz, J₄,5 =5.9, J₄,F =30.3 Hz),3.22 (1H, ddd, H-5, J₄,5 =5.9 Hz, J₅,6a =4.4 Hz, J₅,6b =2.9 Hz ), 2.88(t, 1H, H-6a, J=4.4 Hz), 2.71 (dd, 1H, H-6b, J₅,6b =2.9 Hz, J_(6a),b=4.9 Hz), 1.47, 1.33 (s, each 3H, ipr)

3.82 g (18.7 mmol) of5,6-anhydro-1,2-O-isopropylidene-3-deoxy-3-fluoro-alpha-L-idofuranosewas dissolved in 90 ml of MeOH. To this solution was added 1.42 g (18.7mmol) of thiourea, and the mixture was heated under reflux for 7 hours.After the mixture was allowed to cool, the solvent was distilled off.The residue was partitioned between H₂ O and CHCl₃, and the organiclayer was dried over Na₂ SO₄. The solvent was distilled off to obtain,as a residue (3.99 g),5,6-anhydro-1,2-O-isopropylidene-3-deoxy-3-fluoro-5-thio-alpha-D-glucofuranose.Further, from5,6-anhydro-1,2-O-isopropylidene-3-deoxy-3-fluoro-alpha-L-idofuranose(crude) which was obtained by treating1,2-O-isopropylidene-3-deoxy-3-fluoro-5-O-methanesulfonyl-alpha-D-glucofuranosewith NaH,5,6-anhydro-1,2-O-isopropylidene-3-deoxy-3-fluoro-5-thio-alpha-D-glucofuranosewas obtained as a residue (0.87 g) in the same manner as the above. Thecombined residues were dissolved in a mixture of 15 ml of AcOH and 75 mlof Ac₂ O. To this solution was added 3.46 g (35.29 mmol) of potassiumacetate, and the mixture was heated under reflux for 20 hours. After themixture was allowed to cool, the solvent was distilled off. The residuewas suspended in ethyl acetate, and the suspension was filtered toremove insolubles. The filtrate was sequentially washed with H₂ O×2,saturated NaHCO₃ and brine, dried over Na₂ SO₄, and evaporated todryness under reduced pressure. The residue was purified by columnchromatography on silica gel (220 cc, 15-20% AcOEt (in hexane)). Thus,5.2 g (16.13 mmol) of5,6-di-S,O-acetyl-1,2-O-isopropylidene-5-thio-alpha-D-glucofuranose wasobtained in a yield of 73%.

Melting Point: 88.9-90.4° C.; Elemental Analysis (for C₁₃ H₁₉ O₆ SF);Calculated C: 48.44; H: 5.94; Found C: 48.49; H: 6.00; ¹ H-NMR (CDCl₃) δppm 5.98 (d, 1H, H-1, J₁,2 =3.9 Hz), 4.96 (dd, 1H, H-3, J₃,F =49.6 Hz),4.68 (dd, 1H, H-2, J₁,2 =3.9 Hz), 4.46-4.37 (m, 3H, H-6a, H-6b, H-4),4.11 (dt, 1H, H-5), 2.36 (s, 3H, Ac) 2.06 (s, 3H, Ac), 1.49 (s, 3H,ipr), 1.33 (s, 3H, ipr)

(3) Synthesis of methyl3,5-di-O-benzoyl-2-deoxy-2-fluoro-4-thio-D-arabinofuranose (R₆ =R₇ =Bzand R₈ =Me in formula [VIII])

100 mg (0.31 mmol) of5,6-di-S,O-acetyl-1,2-O-isopropylidene-5-thio-alpha-D-glucofuranose wasdissolved in 90% trifluoroacetic acid (1.5 ml). The solution was stirredat 0° C. for 4 hours, and then diluted with ethyl acetate. The organiclayer was sequentially washed with H₂ O×3, saturated NaHCO₃ ×2 andbrine, and dried over Na₂ SO₄. The solvent was distilled off, and theresidue was dissolved in 0.8 ml of MeOH. To this solution was added 0.8ml of an aqueous solution of 58.4 mg (0.27 mmol) of NaIO₄ at roomtemperature. After the reaction was completed, glycerin was added to thereaction solution, and the mixture was stirred for 30 minutes toterminate the reaction. Insolubles were removed by filtration. Thefiltrate was evaporated to dryness under reduced pressure, and theresidue was partitioned between H₂ O×3 and CHCl₃. The organic layer waswashed with brine, and dried over Na₂ SO₄. The solvent was distilledoff, and the residue was dissolved in 2 ml of 5% HCl/MeOH. The solutionwas heated under reflux for 4 hours, and then neutralized with NaHCO₃.Insolubles were filtered off, and the solvent was distilled off. Theresidue was dissolved in 2 ml of pyridine. To this solution was added150 microlitters (1.29 mmol) of BzCl at 0° C., and the mixture wasstirred at room temperature for 2.5 hours. The reaction was terminatedby adding saturated NaHCO₃, and the reaction solution was extracted withCHCl₃. The organic layer was successively washed with 0.5 N HCl,saturated NaHCO₃ and brine, dried over Na₂ SO₄, and evaporated todryness under reduced pressure. The residue was purified by flash columnchromatography on silica gel (15 cc, 5% AcOEt (in hexane)). Thus, 28 mgof the alpha-anomer, 24.5 mg of the beta-anomer, and 13.1 mg of amixture of the alpha- and beta-anomers were respectively obtained (total54%). (alpha-anomer)

1H-NMR (CDCl₃) δ ppm 8.03-7.99 (4H, m, Bz) 7.60-7.35 (6H, m, Bz), 5.77(1H, dt, H-1) 5.28 (1H, dd, H-2, J₂,F =8.3 Hz), 5.24 (1H, d, H-3, J₂,3=2.0 Hz), 4.61-4.47 (2H, m, H-5a, 5b), 4.05 (1H, dt, H-4), 3.42 (3H, s,OMe) (beta-anomer) ¹ H-NMR (CDCl₃) δ ppm 8.04-8.02 (4H. m, Bz),7.59-7.31 (6H, m, Bz) 6.09 (1H, dt, H-1, J₁,2 =3.9 Hz), 5.35 (1H, ddd,H-2. J₂,F =51.5 Hz) 4.96 (1H, d, H-3), 4.57 (2H, ddd, H-5a, 5b. J_(5a),b=11.2 Hz, J_(5a),4 =J₄,5b =6.4 Hz), 3.69 (1H, dt, H-4, J₄,5a =J₄,5b =6.4Hz), 3.43 (3H, s, OMe)

(4) 1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosine (R=H informula [I])

24.2 mg (0.062 mmol) of methyl3,5-di-O-benzoyl-2-deoxy-2-fluoro-4-thio-alpha- and-beta-D-arabinofuranose was dissolved in a mixture of 2 ml of AcOH and 2ml of Ac₂ O. To this solution was added 0.25 ml of conc. sulfuric acidat 0° C., and the mixture was stirred at room temperature for 1 hour.The mixture was neutralized with 4.5 g of NaOAc, and then partitionedbetween CH₂ Cl₂ and H₂ O. The organic layer was dried over Na₂ SO₄. Thesolvent was distilled off, and the residue was purified by columnchromatography on silica gel (10 cc, 10% AcOEt (in hexane)). Thus, 23.5mg (0.056 mmol) of1-O-acetyl-3,5-di-O-benzoyl-2-deoxy-2-fluoro-4-thio-D-arabinofuranosewas obtained as a mixture of the alpha- and beta-anomers in a yield of91%.

¹ H-NMR (CDCl₃) δ ppm 8.06-7.94 (m, 4H, Bz) 7.62-7.30 (m, 6H, Bz), 6.24(dd, 0.42H, H-1a, J₁,2 =2.0 Hz, J₁,F =14.2 Hz), 6.18 (d, 0.58H, H-1β,J₁,2 =4.4 Hz), 6.08 (ddd, 0.58H, H-3β, J₃,4 =7.3 Hz, J₂,3 =9.3 Hz, J₃,F=11.7 Hz), 5.85 (dt, 0.42H, H-3α, J_(2;3) =J₃,4 =3.9 Hz, J₃,F =12.2 Hz),5.39 (ddd, 0.42H, H-2α, J₁,2 =2.0 Hz, J₂,3 3.9 Hz, J₂,F =47.9 Hz), 5.31(ddd 0.58H, H-2β, J₁,2 =4.4 Hz, J₂.3 =9.3 Hz, J₂,F =50.8 Hz), 4.69 (dd,0.58H, H-5βa, J₄,5a =6.4 Hz , J_(5a),b =11.2 Hz), 4.55 (dd, 0.42H.H-5αa, J₄,5a =7.8 Hz. J_(5a),b =11.7 Hz), 4.49 (dd, 0.58H, H₅ βb, J₄,5b=6.4, J_(5a),b =11.2 Hz 4.47 (dd, 0.42H, H-5αb, J₄,5b =1.5 Hz, J_(5a),b=11.7 Hz) 4.11 (ddd, 0.42H, H-4α, J₃,4 =4.4 Hz, J₄,5 =7.8 Hz, J₄,5b =1.5Hz), 3.74 (q, 0.58H, H-4β, J₃,4 =J₄,5a =J₄,5b =6.4 Hz), 2.12, 2.11 (s,total 3H, Ac)

150 mg (0.358 mmol) of1-O-acetyl-3,5-di-O-benzoyl-2-deoxy-2-fluoro-4-thio-D-arabinofuranosewas dissolved in 1.5 ml of CH₂ Cl₂. To this solution was added 0.3 ml of30% HBr/aetic acid, and the mixture was stirred at room temperature for20 minutes. The reaction was terminated by adding 15 ml of ice water,and the reaction solution was extracted with CH₂ Cl₂. The organic layerwas washed with saturated NaHCO₃ and ice water, dried over Na₂ SO₄, andthen evaporated to dryness at a temperature of 30° C. or lower underreduced pressure to obtain the 1-bromo derivative as an oil. This1-bromo derivative was added to silylated acetylcytosine [prepared bydissolving 82.3 mg (0.538 mmol) of acetylcytosine in 2.5 ml ofdichloroethane, adding 267 microliters (1.08 mmol) of BSA to thesolution, heating the mixture under reflux for 3 hours, andconcentrating the reaction solution to dryness], and glycosylation wasperformed at 80° C. for 5 hours under a reduced pressure of 4 mmHg orlower. The resulting product was suspended in CHCl₃, and the unreactedbase was filtered off. The filtrate was concentrated. The concentratewas dissolved in a mixture of 3 ml of MeOH and 3 ml of NH₄ OH, and thesolution was stirred overnight. The solvent was distilled off, and theresidue was purified by flash column chromatography on silica gel (20cc, CHCl₃ :MeOH (10:1) to (3:1)). Thus, 59.3 mg (0.227 mmol) of1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)-cytosine wasobtained as a mixture of the alpha- and beta-anomers in a yield of63.4%. The beta/alpha ratio determined by HPLC (YMC A-312, 275 nm, 5%CH₃ CN (in 50 mM TEAA)) was 5.02. This mixture was separated into thebeta-anomer and the alpha-anomer in the same manner as in

Synthesis Example 1 Test Example 1

Inhibitory Activity of Proliferation of Cultured Cell (in vitro test)

(Method)

10 microlitters of a sample solution or a Hanks' MEM was placed on a96-well plate. Cells in the logarithmic growth phase were diluted withan RPMI 1640 medium to which 10% of fetal calf serum had been added, andthen inoculated on the plate so that the number of the cells would be5,000/90 microliters/well. This was incubated in a CO₂ -incubator at 37°C. for 3 days. After the incubation was completed, 10 microlitters of anMTT solution (5 mg/ml (in PBS)) was added to each well, and incubationwas conducted in the CO₂ -incubator at 37° C. for a further 4 hours.After the incubation was completed, 100 microlitters of 0.02 Nhydrochloric acid/50% dimethylformamide/20% SDS was added to each well,and the formazane produced was dissolved by stirring. The absorbance at570 nm (test wavelength) or at 690 nm (control wavelength) was measuredby a microplate reader ("Toso MPR 4Ai"). The 50% inhibitoryconcentration (ID₅₀) of the sample was calculated by the probit method,using a computer soft.

In the above test, the test sample was dissolved in dimethyl sulfoxideso that the concentration thereof would be 10 mg/ml; the solution waspreserved at 4° C., and then diluted with a Hanks' MEM; and theresultant was used therein.

(Results)

The results of the test carried out by using the compound of the presentinvention are shown in Table 1. In the table, "CCRF-HSB-2" indicateshuman leukemia cell, "KB" indicates cancer cell of the humanrhinopharynx, and "MKN-45" and "MKN-28" indicate human gastric cancercell.

                  TABLE 1                                                         ______________________________________                                               IC.sub.50 (μg/ml)                                                   Compound CCRF-HSB-2  KB      MKN-45  MKN-28                                   ______________________________________                                        Compound of                                                                            0.051       0.015   2.1     0.027                                    the Invention                                                                 Ara-C    0.052       0.26    >20     0.36                                     ______________________________________                                    

Test Example 2

In Vivo Antitumor Activity on Murine Sarcoma S-180

(Method)

Mouse sarcoma S-180 cells were intraperitoneally subinoculated in ICRmice. 6 or 7 days after the transplantation, abdomen-swollen mice wereselected, and abdominal dropsy was collected therefrom. The abdominaldropsy collected was diluted with sterile PBS so that the number of thecells would be 5×10⁷ /ml. The cell suspension thus prepared wassubcutaneously transplanted to the flanks of female ICR mice of 5 weeksold in an amount of 0.1 ml (5×10⁶ cells) per mouse. A sample wasdissolved in a physiological saline solution (PSS). The resultingsolution was intravenously administered to the mice in an amount of 0.1ml per 10 g body weight once a day for 10 days from the 3rd day afterthe transplantation. On the other hand, the vehicle (PSS) wasadministered to the mice in the control group.

4 weeks after the transplantation, the tumors were removed from themice, and weighed. The antitumor activity was evaluated by comparing theweights of the tumors removed from the mice in the sample administrationgroup with those of the tumors removed from the mice in the controlgroup.

(Results)

The results of the measurement are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                                                     Weight of                                                                     Tumor                                                             Schedule    (average ±                                            Dose     of Admini-  standard  T/C                                    Compound                                                                              (mg/kg)  stration    deviation (%)                                    ______________________________________                                        Control --       Day 3-12    8.20 ± 2.84                                                                          100                                    Compound                                                                              30       Day 3-5, 8-12                                                                             2.09 ± 1.44                                                                          25                                     of the  10       Day 3-12    2.20 ± 0.94                                                                          27                                     Invention                                                                              3       Day 3-12    1.36 ± 0.90                                                                          17                                              1       Day 3-12    5.34 ± 4.11                                                                          65                                     Furtulon                                                                              30       Day 3-12    5.57 ± 1.10                                                                          68                                             10       Day 3-12    9.22 ± 2.90                                                                          112                                    ______________________________________                                    

Test Example 3

In Vivo Antitumor Effect (1) on Human Colonic Carcinoma SW-48 Cells

(Method)

SW-48 cells were cultured in an RPMI 1640 medium containing 10% of fetalcalf serum, harvested in the logarithmic growth phase, andsubcutaneously transplanted to the flanks of female BALB/c nude mice of5 weeks old so that the number of the cells would be 1×10⁷ /mouse. Asample was dissolved in a physiological saline solution (PSS). Theresulting solution was intravenously administered to the mice once a dayfor 10 days (qd×10) from the 7th day after the transplantation. On theother hand, the vehicle (PSS) was dministered to the mice in the controlgroup.

The sizes of -the tumors of the mice were measured twice a week, and thevolumes of the tumors were obtained by calculation from the followingformula. The antitumor effect was evaluated by comparing the growthcurve of the tumors of the mice in the sample administration group withthat of the tumors of the mice in the control group.

    V=(L×W.sup.2)/2

wherein V: the volume of tumor (mm³),

L: the length of tumor (mm), and

W: the width of tumor (mm).

(Results)

The results of the measurement are shown in FIG. 1. In this figure,"Thio-FAC" indicates the compound of the present invention, and "5-FU"indicates 5-fluorouracil.

Test Example 4

In Vivo Antitumor Effect (2) on Human Colonic Carcinoma SW-48 Cells

(Method)

SW-48 cells were cultured in an RPMI 1640 medium containing 10% of fetalcalf serum, harvested in the logarithmic growth phase, andsubcutaneously transplanted to the flanks of female BALB/c nude mice of5 weeks old so that the number of the cells would be 1×10⁷ /mouse.sample was dissolved or suspended in a physiological saline solution(PSS) containing 0.5% carboxymethyl cellulose. The resulting solution orsuspension was orally administered to the mice once a day for theconsecutive 10 days, or every three days four times in all, from the 7thday after the transplantation. On the other hand, the vehicle (PSS usedabove) was administered to the mice in the control group.

The sizes of the tumors of the mice were measured twice a week, and thevolumes of the tumors were obtained by calculation from the aboveformula. The antitumor effect was evaluated by comparing the growthcurve of the tumors of the mice in the sample administration group withthat of the tumors of the mice in the control group.

(Results)

The results of the measurement are shown in FIG. 2. In this figure,"Thio-FAC" indicates the compound of the present invention.

Formulation Example 1: Tablet

    ______________________________________                                        Compound of the invention                                                                        30.0 mg                                                    Cellulose fine powder                                                                            25.0 mg                                                    Lactose            39.5 mg                                                    Starch             40.0 mg                                                    Talc                5.0 mg                                                    Magnesium stearate  0.5 mg                                                    ______________________________________                                    

Tablets are prepared by a conventional method according to the abovecomposition.

Formulation Example 2: Capsule Formulation

    ______________________________________                                        Compound of the invention                                                                        30.0 mg                                                    Lactose            40.0 mg                                                    Starch             15.0 mg                                                    Talc                5.0 mg                                                    ______________________________________                                    

A capsule formulation is prepared by a conventional method according tothe above composition.

Formulation Example 3: Injectable Formulation

    ______________________________________                                        Compound of the invention                                                                         30.0 mg                                                   Glucose            100.0 mg                                                   ______________________________________                                    

An injectable formulation is prepared by dissolving the aboveingredients in purified water for injections.

INDUSTRIAL APPLICABILITY

The compounds of the present invention have excellent antitumoractivity, and are expected to be developed into pharmaceuticals.Further, the production processes of the present invention can bepracticed by using inexpensive materials as starting materials with asmall number of steps and simple operation, so that they are extremelypractical.

We claim:
 1. A1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)-cytosine representedby formula (I): ##STR18## wherein R represents a hydrogen atom or aphosphoric acid residue.
 2. A pharmaceutical composition comprising acompound set forth in claim 1 as the active ingredient, and apharmaceutically acceptable carrier.
 3. A method of treating tumorswhich comprises administering to a patient in need of such treatment,the pharmaceutical composition according to claim
 2. 4. A process forproducing a compound set forth in claim 1, which comprises:1^(st)step:protecting the primary hydroxyl group of compound (II): ##STR19##wherein R₁ and R₂ represent an alkyl, silyl or acyl group; and thenreacting the thus protected compound with diethylaminosulfur trifluoride(DAST) to produce compound (III): ##STR20## .sup. nd step: convertingthe compound of formula (III) into a sulfoxide by reacting it with anoxidizing agent; ##STR21## subjecting the sulfoxide to a Pummererrearrangement reaction by treating it with an acid anhydride or acidchloride, thereby obtaining a compound represented by formula (IV):##STR22## wherein R₁ and R₂ are as defined above, and R₃ represents anacyl group; and .sup. rd step:subjecting the compound represented byformula (IV) and N⁴ -acylcytosine or cytosine to a glycosylationreaction in the presence of a Lewis acid catalyst to obtain a protectedcompound, ##STR23## removing the protecting groups, and, optionally,phosphorylating the 5'-position of the sugar moiety of the compound,thereby obtaining a1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosine representedby formula (I): ##STR24## wherein R₁, R₂ and R₃ are as defined above,and R represents a hydrogen atom or a phosphoric acid residue; andrecovering compound (I).
 5. A process for producing a compound set forthin claim 1, which comprises:1^(st) step:a step of introducing a leavinggroup into the hydroxyl group on a compound represented by formula (V),##STR25## and treating the compound with a nucleophilic reagent by whicha fluorine atom can be introduced, thereby obtaining a compoundrepresented by formula (VI): ##STR26## 2^(nd) step: a step ofselectively deprotecting the isopropylidene group at the 5- and6-positions of the compound represented by formula (VI): ##STR27##selectively protecting the primary hydroxyl group of the compound,##STR28## introducing a leaving group into the secondary hydroxyl groupof the compound, ##STR29## deprotecting the primary hydroxyl group andperforming 5,6-epoxidation, ##STR30## performing 5,6-thiiranation byusing a sulfurizing reagent, ##STR31## opening the thiirane ring byusing a nucleophilic reagent, and causing acylation thereby obtaining acompound represented by formula (VII): ##STR32## wherein R₄ and R₅represent an alkyl or acyl group; .sup. rd step: hydrolyzingisopropylidene group at the 1- and 2-positions of the compoundrepresented by formula (VII), ##STR33## carrying out oxidation by usingan oxidizing agent, ##STR34## alkoxylating the 1-position, ##STR35## andprotecting the hydroxyl group with a protecting group, thereby obtaininga compound represented by formula (VIII): ##STR36## wherein R₄ and R₅are defined as above, R₆ and R₇ represent an alkyl or acyl group, and R₈represents an alkyl group; and .sup. th step:brominating the alkoxygroup at the 1-position of the compound represented by formula (VIII) bytreating the compound (VIII) with a hydrogen bromide-acetic acidsolution, ##STR37## subjecting the brominated compound and activatedcytosine to glycosylation reaction to obtain a protected compound,##STR38## removing the protecting groups, and optionally,phosphorylating the 5'-position of the sugar moiety of the compound,thereby obtaining a1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosine representedby formula (I): ##STR39## wherein R₆, R₇, and R₈ are as defined above,and R represents a hydrogen atom or a phosphoric acid residue, andrecovering compound (I).